Screening for potential target genes for RNAi mediated mosquito control

To control mosquitoes by using the data from analysis of transcriptome, total of nine potential RNAi targets genes were selected because these genes showed depleted by both loreclezole hydrochloride and K21877 treatment. To validate the analysis of differentially expressed genes related potential RNAi targets from the RNA-seq data, the expression level of these genes were calculated by qPCR (Fig. 33 and 34). As shown in the Figures, the qPCR results confirmed the RNA-seq data, with all analyzed genes down-regulated by loreclezole hydrochloride and K21877 treated.

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Figure 33. Validation of the Illumina sequencing result of the potential target genes for RNAi by qPCR.

The relative transcription levels were demonstrated in log10 scale of K222212-treated to control sample.

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Figure 34. Validation of the Illumina sequencing result of the potential target genes for RNAi by qPCR.

The relative transcription levels were demonstrated in log10 scale of K21877-treated to control sample.

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DISCUSSION

Technological advancement of sequencing could have allowed large-scale comparative studies to be performed that were unimaginable in the past. Sequencing technologies are various methods that are grouped broadly as template preparation, sequencing and imaging, and data analysis. The variety of sequencing platforms is produced by the combination of sequencing methods (Metzker, 2010; Simon et al., 2009). In this chapter, the transcript level responses of A. albopictus treated with JHA or JHAN were analyzed by Illumina sequencing and bioinformatics methods because the Illumina sequencing is one of most widely used.

Although the transcripts of A. albopictus are available, the transcripts of A. aegypti are used by the reference gene sequences to analyze the response to JH-related IGRs in A.

albopictus because not only genomics and transcriptomics studies, but also

endocrinological studies including JH are better studied in A. aegypti than that of A.

albopictus.

In 1996, the genome of Saccharomyces cerevisiae was completely sequenced. After then, the complete genomic sequences of higher model organism including human were announced. The comparison between complete eukaryotic genomes revealed that biological roles of numerous genes and proteins could be inferred from their similarity to their putative orthologues. This high degree of sequence and functional conservation among eukaryotes has led to unification of biological annotations. The Gene Ontology (GO) Consortium was established to maintain and develop a controlled and

organism-104

independent vocabulary for describing the roles of genes and gene products. GO maintains vocabularies of three domains, Biological process (GO:0008150, BP or P), Molecular function (GO:0008639, MF or F), and Cellular component (GO:0005575, CC or C). These three categories of GO represents information sets that are common to all significance to over/under-represented GO terms between groups. Therefore, the RNA-seq data which mapped to the transcripts of A. aegypti were performed GO enrichment analysis in order to identify GO terms affected by JHA or JHAN treatments. The GO enrichment analysis showed that 35, 6, and 12 GO terms were significantly enriched by loreclezole hydrochloride and depleted by K21877 treated in Biological process, Molecular function, and Cellular component, respectively.

JH is highly versatile hormone and immune responses are also regulated by JH. 20-hydroxy-ecdysone (20E) is commonly known to induce expression of AMP genes, such as diptericin and drosomycin, and JH strongly interfere with 20E-dependent immune response in Drosophila melanogaster (Dimarcq et al., 1997; Flatt et al., 2008; Meister and Richards, 1996; Silverman et al., 2000). In contrast, several AMP genes, including drosocin and defensin, were down regulated by 20E in EcR-dependent manners

(Beckstead et al., 2005). The conflicting reports in Drosophila imply that JH and 20E regulate expression of AMP genes in a complex manner. However, how these hormones

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regulate the immune responses remains unclear. Especially, there is a lack of understanding about the roles of JH or JH-dependent interaction of Met in immune responses. In this study, total of 275 genes were categorized into 27 immune-related gene families. Among them, differential expressions of two AMP genes, defensin (AAEL003857-RA) and cecropin (AAEL000621-RA), were noteworthy (Fig. 35).

Defensin was up and down-regulated by loreclezole hydrochloride (JHA) and K21877

(JHAN), respectively, while cecropin was regulated vice-versa. These results need to consider aspect of the spectrum of AMPs. AMPs are an important response of the humoral immunity and seven AMP families exist in Drosophila (Lemaitre and Hoffmann, 2007). However, only three of them were identified in mosquitoes, including defensins, cecropins and attacins. Among them, AMPs of mosquito are mainly represented by defensins and cecropins (Waterhouse et al., 2007). Generally, mosquito defensins are active against Gram-positive bacteria and cecropins have a broad spectrum of antimicrobial activity (Kokoza et al., 2010; Lowenberger et al., 1995; Lowenberger et al., 1999; Vizioli et al., 2000). Consequently, these results suggested that AMPs that have a broad-spectrum could be expressed by low JH titer in hemolymph because JH titer is generally being maintained at low level and, in contrast, expression of several AMPs could be regulated by JH against type of pathogenic bacteria.

All aerobic organisms need to respire in order to use molecular oxygen, and toxic reactive oxygen species (ROS) such as superoxide radical (O·2

-), hydrogen peroxide (H2O2) and hydroxyl radical (·OH) are generated during the reduction of molecular oxygen to water (Halliwell and Gutteridge, 2015). To detoxify these reactive free radicals, various defense systems are used (Hogg and Kalyanaraman, 1999). Peroxidases-related

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immune response is important to detoxify ROS, and the peroxidases-related genes are regulated by JH and 20E (Tarhan et al., 2013). In this study, RNA-seq data and qPCR result showed that peroxidases-related genes, AAEL000795-RA (glutathione peroxidase) and AAEL005416-RA (oxidase/peroxidase), were up-regulated by loreclezole hydrochloride and down-regulated by K21877 (Fig. 35). In these results, expression of AMP genes and peroxidases-related genes are not only related with JH activity but also JH-dependent interaction of Met. These results demonstrated that hormonal regulation, including JH, of immune response is a complicated process.

Figure 35. Validation of the Illumina sequencing result of immune-related genes by qPCR. The relative transcription levels were demonstrated in log10 scale of IGR-treated to control sample.

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Among JH-regulated biological activities in insects, the role of transcription factors is most important for regulating various physiological functions. Through the regulation of genes acting downstream of JH, JH could play a key role in insects. It have been reported that JH-dependent interaction of Met promoted ribosomal biogenesis through expression of gene encoding the regulator of ribosomal synthesis 1 (RRS1) and six ribosomal protein in A. aegypti (Wang et al., 2017a). This result suggested that JH controls the proliferation of ribosomes to sustain an enhanced translation rate. In this study, Ribosome biogenesis in eukaryotes (KEGG reference pathway: map03008) and RNA transport (KEGG reference pathway: map03013) related genes significantly enriched by loreclezole hydrochloride -treatment. Among 90 reference genes of A. aegypti related to the Ribosome biogenesis in eukaryotes pathway, 55 genes were identified in the RNA-seq results. Among them, total of 32 genes (58%) were enriched and depleted by loreclezole hydrochloride and K21877 treatment, respectively (Fig. 36). And then, among 146 reference genes of A. aegypti related to the RNA transport pathway, 106 genes were identified in the RNA-seq results. Likewise, total of 61 genes (57%) were enriched and depleted by loreclezole hydrochloride and K21877 treatment, respectively (Fig. 37). As shown in the Fig. 38, the qPCR results were in good accordance with the RNA-seq results (Fig. 38). These results suggested that JH is critical in translation for inducing the expression of genes that related to Ribosome biogenesis in eukaryotes and RNA transport pathway, and the genes related to these pathways were enriched and depleted by loreclezole hydrochloride and K21877 treatment, respectively. The overall results of differentially expressed gene analysis based on GO annotation showed that transcription levels were enriched by JHA and JHAN compound treatment, respectively. These results

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can be inferred from relationship between JH-dependent interaction of Met and the translation.

Figure 36. Analysis of differentially expressed genes related to the Ribosome biogenesis in eukaryotes pathway. 55 genes related to the pathway were assigned based on the expression pattern by treatment of K222212 and K21877. L_up and K_up represent the up-regulated genes by K222212 (L) and K21877 (K), respectively. L_down and K_down also represent the down-regulated genes by K222212 (L) and K21877 (K), respectively.

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Figure 37. Analysis of differentially expressed genes related to the RNA transport pathway. 106 genes related to the pathway were assigned based on the expression pattern by treatment of K222212 and K21877.

L_up and K_up represent the up-regulated genes by K222212 (L) and K21877 (K), respectively. L_down and K_down also represent the down-regulated genes by K222212 (L) and K21877 (K), respectively.

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Figure 38. Validation of the Illumina sequencing result of translation-related genes by qPCR. The relative transcription levels were demonstrated in log10 scale of IGR-treated to control sample.

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Aedes mosquitoes have shown a remarkable ability to develop resistance to insecticides.

Therefore, resistance to commercially available insecticides, such as pyrethroids, is widespread in Aedes mosquitoes (Brown, 1986). In this study, novel JHA and JHAN compounds were identified by using yeast two-hybrid β-galactosidase binding assay and these compounds were characterized as in vivo modulation of JH-regulated physiological functions such as expression of JH-responsive genes and vitellogenesis. However, it could be possible that the JHA and/or JHAN compounds may also have cytotoxicity to Aedes mosquitoes. Therefore, the differential expressions of the genes which are related

to detoxification of pyrethroids, resistance strain of A. aegypti, were selected. In RNA-seq data, 17 and seven of those genes were up- and down-regulated by loreclezole hydrochloride and K21877 treatment, respectively. Especially, cytochrome p450 CYP9J27 v2 (AAEL014607-RA) gene is the most typical gene in CYP9 family of

Cytochrome P450 genes that over-expressed in pyrethroids resistance strain of A. aegypti (Ishak et al., 2017) and this gene was validated by qPCR. These results demonstrated that both JHA and JHAN have cytotoxicity to A. albopictus and this cytotoxicity induces the genes related to detoxification of pyrethroids in Aedes mosquito. This cytotoxicity is important advantages to control mosquito. The first advantage is that both loreclezole hydrochloride and K21877 have not only general advantage of JH-related IGR, but also comparatively rapid mosquitocidal activity. This is important to overcome the disadvantage that JH-related IGR do not cause immediate knockdown. Another advantage is the possibility that those compounds could use for control of resistance mosquitoes to commonly used insecticides.

Nearly 20 years ago, the application of exogenous dsRNA to interfere with translation

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of the homolog endogenous mRNA was reported and called this process RNA interference (RNAi) (Fire et al., 1998). This technique was described as ‘post-transcriptional gene silencing’ (Fire, 2007). RNAi soon proved to be useful in several research fields such as gene function determination in genomics. In agriculture, it shows great potential for pest control because of its high specificity (Gordon and Waterhouse, 2007; Price and Gatehouse, 2008). A new approach to control mosquitoes using the RNA-seq data from A. albopictus treated with exogenous JHA or JHAN was tried. If the candidate genes for RNAi target are selected, the genes could be essential for survival of the mosquito. For a new approach, nine genes were selected, and the proteins that coded by the candidate genes have essential functions such as metabolism-related (AAEL004042-RB; probable glucosamine 6-phosphate N-acetyltransferase, AAEL000321-RA; acetyl-coenzyme A synthetase, AAEL007653-RA; allantoinase), signal transduction-related (AAEL012001-RA; Anopheles gambiae PEST galectin) and structure-related (AAEL013789-RA; cuticle-like, AAEL007745-RA; blood vessel epicardial, AAEL002191-RA; adult cuticle 1-like, AAEL002185-RA; cuticle, AAEL013139-RG; cytokinesis actomyosin contractile ring assembly). To identify availability of the candidate genes for RNAi, expression level of those genes were calculated by qPCR. The qPCR result demonstrated that the transcription levels of those genes were down-regulated by exogenous JHA and JHAN, and the results were in good accordance with the sequence analysis result. If mosquitocidal activities of dsRNAs of those genes are proven through further studies, the processing of dsRNA for silencing of the genes could be as effective as the processing of loreclezole hydrochloride or K21877 in mosquito. These results could provide important genetic information about the RNAi

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In conclusion, to investigate the transcriptional responses of the mosquito treated with JHA and JHAN, comprehensive transcriptome sequencing of adult female of A.

albopictus topically applied with loreclezole hydrochloride and K21877 was performed.

Total of 56 GO terms were up- and down-regulated by loreclezole hydrochloride and K21877 treatment, respectively. Also, through the modulation of translation-related genes, such as the genes that related to Ribosome biogenesis in eukaryotes and RNA transport pathway, the relationship between JH-dependent interaction of Met and the translation were identified. The results from investigation of the effects of JH on differential expression of immune-related genes were that the genes related to several AMPs and peroxidases were regulated by JHA and JHAN. Furthermore, new approaches to identify cytotoxicity of loreclezole hydrochloride and K21877 and to select candidate genes for mosquito control using dsRNA-based JH-responsive genes provided a clue to overcome the disadvantage that JH-related IGR and information for mosquito control used dsRNA.

Therefore, these results will provide important information for the transcriptional responses treated with JHA and JHAN in mosquito.

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GENERAL DISCUSSION

Among various insects, several insects need to be controlled because they not only cause severe economic damage to agricultural products but also act as vectors of human diseases (Boyer et al., 2012; Hill et al., 2005). Chemical insecticides have been commonly used to minimize the economic losses and to protect public health. Especially, among the commonly used chemical insecticides, neuroactive insecticides are effective to protect crops, agricultural products, livestock, and people. The major nerve targeting insecticides such as organophosphates, methylcarbamates, neonicotinoids, and pyrethroids act rapidly to stop crop and agricultural product damage and disease transmission. However due to their toxicity to the environment, safety issues to non-target organisms, and the development of resistance, sustained use of these insecticides are limited (Casida and Durkin, 2013). Actually, they have a limited scope of application when insecticides are used to control of urban pests, household pests or their resistant pests. Therefore, there are growing demands for novel insecticides which are more effective and safe than currently used insecticides.

IGRs can be highly potent, effective, and selective insecticides and are enough alternatives to overcome the limitation of using chemical insecticides for pest control.

Among types of IGRs, based on non-target insect and safety tests conducted in industry, government, and academic laboratories, JHAs appear to be safer insecticides when compared to the neurotoxic insecticides used in pest management. Various JHAs such as methoprene, fenoxycarb, and pyriproxyfen have been used for control of several insects

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in field. However, use and development of JHA insecticides also were limited because there are several recent reports about the resistance to JHAs and the lack of understanding of mode of actions of JHAs at the molecular level.

Therefore, there is an obvious need for more studies about identification and development of novel JH-related IGR insecticides which have a new structure and a new mode of action of JH-related IGRs. For these studies, Aedes mosquitoes are the most suitable target insects with many reasons. Firstly, Aedes mosquitoes are major vector insects causing public health problems became a biggest threat to many people, therefore the mosquito control is prerequisite to protect public health. Among Aedes mosquitoes, A.

aegypti and A. albopictus are aggressive pests and efficient disease vectors. A. aegypti is

the main vector of important arboviruses including yellow fever, dengue, and chikungunya virus and A. albopictus also transmits devastating pathogens and parasites including malaria, yellow fever, dengue, West Nile, chikungunya, and zika virus (Benelli, 2015; Grard et al., 2014; Gurugama et al., 2010; Reiter et al., 2006; Sang et al., 2015;

Tabachnick, 1991; Weaver, 2014). It is also important to choose the proper insecticides for control of mosquito because these mosquitoes are the most important household and urban pests. Secondly, various JHAs have already been used mosquito control.

Methoprene and pyriproxyfen have been extensively tested against Aedes spp. and shown to be highly effective, both under laboratory and under field conditions (Ramaseshadri et al., 2012). The World Health Organization has even approved the use of methoprene in drinking water for control of mosquitoes (Glare and O'Callaghan, 1999). It means that JH-related IGR insecticides are more effective and safer alternatives than currently used chemical insecticides to control mosquitoes. Finally, the molecular mechanism of JH,

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based on a receptor mediated mode of action, is relatively well established. Since the identification of a JH receptor from Drosophila, there have been many studies on the interaction between JH and its receptor in Drosophila, Tribolium, and Aedes. Especially in Aedes, through a yeast two-hybrid screen, several bHLH-PAS proteins were identified as partner of Met and high-throughput screening of JHAs and JHANs could be performed (Lee et al., 2015; Li et al., 2011; Shin et al., 2012).

In this study, the yeast two-hybrid β-galactosidase assays using the yeast cells transformed with the genes of JH receptor and its partner, Met-FISC of A. aegypti were performed to identify novel JHA and JHAN compounds. Through the screening, loreclezole hydrochloride and penfluridol were isolated based on their availability for the strength of JHA and JHAN activity, respectively. These compounds also showed high level of insecticidal activity against 3rd instar larvae of A. albopictus. Further studies on the insecticidal activity against A. albopictus treated with both JHA and JHAN compounds at the same time would provide important information about its field application.

To find more effective compounds with JH-related IGRs activity for control of mosquitoes, loreclezole hydrochloride and its derivatives were synthesized and their biological characteristics were investigated. Among these derivatives, K21877 demonstrated high level of JHAN activity. Although both loreclezole hydrochloride and K21877 have similar structures, one compound simulated the binding of A. aegypti Met-FISC while the other interfered with the pyriproxyfen-mediated binding of A. aegypti Met-FISC. Both the JHA and JHAN demonstrated high larvicidal activities, embryonic lethality and adult toxicity against A. albopictus, which were due to the modulation of

JH-117

regulated physiological functions such as expression of JH-responsive genes and follicle development. If more derivatives of loreclezole hydrochloride will be synthesized, it would provide greater insight into the relationship between chemical structure and JH-related IGR activity through the screening system.

To investigate the transcriptional responses of the A. albopictus treated with loreclezole hydrochloride and K21877, comprehensive transcriptome sequencing was performed and analyzed. These results suggested that JH-interaction of Met could regulate the expression of genes that related metabolic process, nucleotide binding process, and translation pathway and loreclezole hydrochloride and K21877 could modulate the JH-regulated gene expression. Although the various analyses of related immune, detoxification, and RNAi target suggested the possibility of application of JH-related IGR with currently used chemical and biological insecticides, further studies of molecular and biological aspects need to be designed to provide more information.

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